The present invention relates to human diacylglycerol kinase proteins (hDAGK) and particularly to human diacylglycerol kinase xcex2 (hDAGKxcex2) protein, and to related nucleotide sequences, expression vectors, cell lines, antibodies, screening methods, compounds, methods of production and methods of treatment, as well as other related aspects.
Diacylglycerol kinases (DAGKs) are a family of enzymes that convert diacylglycerol (DAG) to phosphatidic acid and are therefore known to attenuate DAG-dependent protein kinase C activation (PCK) (1).
Five types of DAGKs have been described. Type I DAGKs contains four conserved regions, the N-terminal region (C 1), two sets of EF-hand motifs (C2), two cysteine-rich zinc finger like structures (C3) and the C-terminal region (C4). Type II isoenzymes contain a pleckstrin homology (PH) domain at the N-terminus. Type Ill contains only the zinc finger (C3) and the catalytic region (C4). Type IV contains four ankyrin repeats near the carboxyl terminus. Type V contains three instead of two zinc finger structures, a proline-rich region and a PH domain with an overlapping Ras-associating (RA) domain. All share two domains, the C2 and the C3 (2).
A 90 kDa DAGKxcex2 (3) belonging to type I was found by screening a rat brain cDNA library using fragments of rat a DAGK cDNA under low stringency conditions. The cDNA clone obtained was completely sequenced. The rat DAGKxcex2 cDNA has an open reading frame of 5927 nucleotides and encodes for a protein of 801 amino acids with a predicted relative molecular mass of 90,000. Analysis of the amino acid sequence identified 2 EF-hand motifs (aa 152-180 and 197-225), of two cysteine-rich zinc-finger-like sequences (aa 257-292 and 319-356), and putative ATP-binding sites (aa 266-294 and 533-560).
Brain expression has been described for the known DAGK isoforms (2), which include DAGKxcex1, DAGKxcex7, DAGKxcex6 and DAGKxcex8. A particular distribution restricted to specific regions of the central nervous system (CNS) was described for the DAGKxcex2 form, originally identified in the rat (90 Da DAGKxcex2). The rat beta form is predominantly localised in neurons of the caudate-putamen, the accubens nucleus and the olfactory tubercle. Such brain regions are among the main CNS dopaminergic, serotonergic, acetylcholinergic and glutamatergic terminal fields (4).
It has also been demonstrated that some metabotropic dopamine, serotonin, glutamate, acetylcholine and several peptide receptors are coupled with the phosphoinositide signal transduction system (5). Lithium is known as one of the most effective therapies for bipolar disorders. Although the biological mechanisms of the mood stabilising properties of lithium are not well understood, experimental evidence indicates that lithium modulates the phosphoinositide signal transduction system (6) by inhibiting the phosphatase that liberates inositol from inositol phosphate (IP), and by modifying the activity of the phospholipase C (PLC)-dependent signalling pathways, including the levels of the second messenger diacylglycerol (DAG) that activates protein kinase C (PKC).
With this background in mind, the present inventors have determined that the DAGKs, and particularly the human ortholog of rat 90 kDa DAGKxcex2 (hDAGKxcex2) and variants thereof will provide targets for the development of novel mood stabilising agents and therapeutic agents for treatment of other disorders.
Clearly there is a need for proteins and related nucleotide sequences that may be used to screen for mood stabilising agents and which may also play a role in preventing, ameliorating or correcting mood disorders and dysfunction and other neurological diseases.
Accordingly, it is an object of the present invention to identify targets for screening of novel therapeutic agents. It is another object of the invention to locate and characterise human DAGKxcex2 and variants thereof. Other objects of the present invention will become apparent from the following detailed description thereof.
According to one embodiment of the present invention there is provided an isolated human diacylglycerol kinase xcex2 (hDAGKxcex2) protein or a variant thereof. In a particularly preferred embodiment of this invention the human diacylglycerol kinase xcex2 (hDAGKxcex2) has the amino acid sequence set out in Seq ID No 1 or Seq ID No 4. The hDAGKxcex2 protein having the amino acid sequence set out in Seq ID No 4 is referred to as SV-hDAGKxcex2 protein.
According to one embodiment of the present invention there is provided a nucleotide sequence encoding a human diacylglycerol kinase xcex2 (hDAGKxcex2) protein or a variant thereof, or a nucleotide sequence that is complementary thereto. In a particular preferred embodiment of the invention the polynucleotide comprises the sequence set out in Seq ID No 3 or Seq ID No 6. The hDAGKxcex2 protein having the polinucletide sequence set out in Seq ID No 6 is referred to as SV-hDAGKxcex2 protein.
In accordance with another aspect of the invention there is provided an expression vector comprising a nucleic acid sequence as referred to above which is capable of expressing a hDAGKxcex2 protein.
According to a further aspect of the invention there is provided a stable cell line comprising an expression vector as referred to above. Preferably the cell line is a modified HEK293T, CHO, HeLa, Sf9 or COS cell line.
According to yet a further aspect of the invention there is provided an antibody specific for a hDAGKxcex2 protein.
According to still another aspect of the invention there is provided a method for identification of a compound that exhibits DAGK modulating activity, comprising contacting a DAGK protein with a test compound and detecting modulation of enzyme activity or detecting enzyme inactivity. Preferably the DAGK is hDAGKxcex2 or a variant thereof.
According to another aspect of the invention there is provided a compound which modulates hDAGK activity, identifiable by the method referred to above. Preferred compounds according to the present invention are those that modulate hDAGKxcex2 activity or a variant thereof.
According to another aspect of the invention there is provided a compound that modulates hDAGK activity. Preferred compounds according to the present invention are those that modulate hDAGKxcex2 activity or a variant thereof.
According to a further aspect of the invention there is provided a method of treatment or prophylaxis of a disorder that is responsive to modulation of hDAGK activity in a human patient, which comprises administering to said patient an effective amount of a compound as referred to above. Conveniently the hDAGK is hDAGKxcex2 or a variant thereof. Preferably the disorder is a mood disorder, epilepsy, a neurodegenerative disorder, anxiety, schizophrenia, migraine, drug dependence, stroke, Alzheimer""s dementia or Parkinson""s disease.
According to a further aspect of the invention there is provided a method of treatment or prophylaxis of a disorder that is responsive to modulation of hDAGK activity in a human patient which comprises administering to said patient an effective amount of a modulator of hDAGK activity. Conveniently the hDAGK is hDAGKxcex2 or a variant thereof. Preferably the disorder is a mood disorder, epilepsy, a neurodegenerative disorder, anxiety, schizophrenia, migraine, drug dependence, stroke, Alzheimer""s dementia or Parkinson""s disease.
According to another aspect of the invention there is provided use of a compound as referred to above in a method of formulating a medicament for treatment or prophylaxis of a disorder that is responsive to modulation of hDAGK activity in a human patient. Conveniently the hDAGK is hDAGKxcex2 or a variant thereof. Preferably the disorder is a mood disorder, epilepsy, a neurodegenerative disorder, anxiety, schizophrenia, migraine, drug dependence, stroke, Alzheimer""s dementia or Parkinson""s disease.
According to another aspect of the invention there is provided use of a modulator of hDAGK activity in a method of formulating a medicament for treatment or prophylaxis of a disorder that is responsive to modulation of hDAGK activity in a human patient. Conveniently the hDAGK is hDAGKxcex2 or a variant thereof. Preferably the disorder is a mood disorder, epilepsy, a neurodegenerative disorder, anxiety, schizophrenia, migraine, drug dependence, stroke, Alzheimer""s dementia or Parkinson""s disease.
According to another aspect of the invention there is provided a method of producing a hDAGKxcex2 protein or a variant thereof comprising introducing into an appropriate cell line a suitable vector comprising a nucleotide sequence encoding for a hDAGKxcex2 protein or a variant thereof, under conditions suitable for obtaining expression of the hDAGKxcex2 protein or variant.
Seq ID No 3 shows the complete nucleotide sequence of the human DAGKxcex2. Seq ID No 5 shows the complete nucleotide sequence of the SV-hDAGKxcex2.
Seq ID No 1 shows the nucleotide and encoded amino acid sequence of the human DAGKxcex2 sequence. Seq ID No 4 shows the nucleotide and encoded amino acid sequence of the SV-hDAGKxcex2.
Seq ID No 7 shows pairwise alignment of hDAGKxcex2 and SV-hDAGKxcex2 full-length amino acids sequences with rat homologue.